Many diseases and injuries of the central nervous system presently are not treatable. These diseases and injuries are untreatable because, unlike many peripheral organs that can be removed in whole or in part, or transplanted, the central nervous system has very limited regeneration capability and cannot be totally excised without death. The eye is an extension of the brain, and therefore a part of the central nervous system. Accordingly, in the case of an eye injury or disease, i.e., a retinal injury or disease, the diseases are often without treatment and the eye cannot be transplanted. Eye diseases and injuries that presently are untreatable include retinal photic injury, retinal ischemia-induced eye injury, age-related macular degeneration, and other eye diseases and injuries that are induced by free radical species.
It has been hypothesized that a major cause of these untreatable central nervous system diseases and injuries is the generation and presence of singlet oxygen and other free radical species. Singlet oxygen and free radical species can be generated by a combination of light and oxygen, or during reperfusion after an ischemic insult.
The eye is subjected to continuous light exposure because the primary purpose of the eye is light perception. Therefore, some untreatable diseases and injuries to the eye result from the continuous exposure of the eye to light, coupled with the highly-oxygenated environment in the eye.
The process of light perception is initiated in the photoreceptor cells. The photoreceptor cells are a constituent of the outer neuronal layer of the retina, which is a component of the central nervous system. The photoreceptor cells are well sheltered in the center of the eye, and are protected structurally by the sclera, nourished by the highly-vascularized uvea and safeguarded by the blood-retinal barrier of the retinal pigment epithelium.
The primary function of the photoreceptor cells is to convert light into a physio-chemical signal (transduction) and to transmit this signal to the other neurons (transmission). During the transduction and transmission processes, the metabolic activities of these neurons are changed dramatically. Even though the photoreceptor cells are securely protected in the interior of the eye, these cells are readily accessible to light because their primary function is light detection. Excessive light energy reaching the retina can cause damage to these neurons, either directly or indirectly, by overwhelming the metabolic systems of these cells.
The combination of continuous and/or excessive exposure to light, and the relatively high concentration of oxygen in the eye, generates singlet oxygen and other free radical species. Singlet oxygen and free radical species also can be generated by enzymatic processes independent from light exposure. The free radical species and singlet oxygen are reactive entities that can oxidize polyunsaturated fatty acids. The retina contains the highest concentration of polyunsaturated fatty acids of any tissue in the human body, and peroxidation of the polyunsaturated fatty acids in cell membranes of the retina by hydroxyl radicals (OH) or superoxide (O.sub.2) radicals can propagate additional free radical species. These free radical species can lead to functional impairment of the cell membranes and cause temporary or permanent damage to retinal tissue. It has been theorized that the generation of singlet oxygen and free radical species therefore underlies the pathogenesis of light-induced retinopathy and post-ischemic reflow injury (i.e., free radical generation during reperfusion after an ischemic insult). In addition, a deficiency in removing these species can also contribute to various diseases of the eye and the central nervous system.
A number of natural mechanisms protect the photoreceptor cells from light injury. For example, the ocular media, including the cornea, aqueous, lens, and vitreous, filter most of the light in the ultraviolet region. However, after cataract extraction or other surgical intervention, some of these protective barriers are removed or disturbed, whereby the photoreceptor cells are more susceptible to damage by radiant energy. The photoreceptor cells also possess other forms of protection from photic injury, for example, the presence of antioxidant compounds to counteract the free radical species generated by light. As will be demonstrated hereafter, antioxidants, which quench and/or scavenge singlet oxygen, hydrogen peroxide, superoxide and radical species, help minimize injury to the photoreceptor cells. In addition, the human eye has an excessive number of photoreceptor cells such that only destruction of a significant number of photoreceptor cells adversely affects visual function.
Even though several protective mechanisms are present in the eye, a leading cause of blindness in the United States is age-related photoreceptor degeneration. Clinically, photoreceptor degeneration, as seen in age-related macular degeneration, is causally related to excessive exposure to blue light. The causes of age-related macular degeneration, which is characterized by a loss of photoreceptor neurons resulting in decreased vision, are being investigated. Epidemiological studies indicate that age-related photoreceptor degeneration, or age-related macular degeneration, is caused by several factors including age, sex, family history, color of the iris, nutritional deficiency, immunologic disorders, cardiovascular and respiratory diseases and preexisting eye diseases. Advancing age is the most significant factor. Recently, it has been demonstrated that aging eyes have a decreased amount of carotenoids. Clinical and laboratory studies indicate that photic injury is a cause of age-related macular degeneration because of the cumulative effect of repeated mild photic insult which leads to a gradual loss of photoreceptor cells.
Age-related macular degeneration is an irreversible blinding disease of the retina. Unlike cataract wherein vision can be restored by replacing the diseased lens, age-related macular degeneration cannot be treated by replacing the diseased retina because the retina is a component of the central nervous system. Therefore, because no treatment for this disease exists once the photoreceptors are destroyed, prevention is the only way to confront age-related macular degeneration. Presently, prevention of age-related macular degeneration resides in limiting or preventing light and oxygen-induced (i.e., free radical-induced) damage to the retina because the retina is the only organ that is continuously exposed to high levels of light in a highly-oxygenated environment.
In addition to photic injury, eye injury and disease can result from singlet oxygen and free radical species generated during reperfusion after an ischemic insult. Ischemic insult to retinal ganglion cells and to neurons of the inner layers of retina causes loss of vision. Loss of vision accompanies diabetic retinopathy, retinal arterial occlusion, retinal venous occlusion and glaucoma, each of which ischemically insults the eye, i.e., deprives the eye of oxygen and nutrition.
The damage to the retinal ganglion cells has been attributed to ischemia, and subsequent reperfusion during which free radicals are generated. During reperfusion, the reoxygenation of tissue after an ischemic insult results in a relatively high concentration of oxygen flowing through the effected tissue, which contributes to free radical formation. Ischemic insult and reperfusion accompanied by free radical generation also is a major contributor to central nervous system damage, such as damage caused by a stroke.
The pathogenesis of photic injury, of age-related macular degeneration, of ischemia/reperfusion damage, of traumatic injury and of inflammations of the eye and central nervous system have been attributed to singlet oxygen and free radical generation, and subsequent free radical-initiated reactions. Investigators therefore studied the role of antioxidants in preventing or ameliorating these diseases and injuries of the central nervous system in general, and the eye in particular.
For example, ascorbate was investigated as an agent to treat retinal photic injury. Ascorbate is a reducing agent which is present in the retina in a high concentration. Studies indicated that ascorbate in the retina can act as an antioxidant and is oxidized by free radical species generated during excessive light exposure.
Administration of ascorbate reduced the loss of rhodopsin after photic exposure, thereby suggesting that ascorbate offered protection against retinal photic injury. A decrease in rhodopsin levels is an indicator of photic eye injury. The protective effect of ascorbate is dose-dependent, and ascorbate was effective when administered before light exposure. Morphometric studies of the photoreceptor nuclei remaining in the retina after light exposure showed that rats given ascorbate supplements had substantially less retinal damage. Morphologically, rats with ascorbate supplements also showed better preservation of retinal pigment epithelium.
The above studies led to the hypothesis that ascorbate mitigates retinal photic injury because of its antioxidant properties, which are attributed to its redox properties. Ascorbate is a scavenger of superoxide radicals and hydroxy radicals and also quenches singlet oxygen and reduces hydrogen peroxide, all of which are formed in retinal photic injury. This hypothesis accounts for the presence of high levels of naturally-occurring ascorbate in a normal retina.
Therefore, antioxidants which inhibit free radical formation, or which quench singlet oxygen and scavenge for free radical species, can decrease lipid peroxidation and ameliorate photic injury and ischemic/reperfusion injury in the central nervous system, and particularly in the retina. Antioxidants originally were investigated because they are known constituents of human tissue. However, antioxidants that are not naturally occurring in human tissue also were tested. In particular, in addition to ascorbate, antioxidants such as 2,6-di-tert-butylphenol, .gamma.-oryzanol, .alpha.-tocopherol, mannitol, reduced glutathione, and various carotenoids have been studied for an ability to quench singlet oxygen and scavenge free radical species. These and other antioxidants are effective quenchers and scavengers for singlet oxygen and free radicals. In particular, the carotenoids, as a class of compounds, are very effective singlet oxygen quenchers and free radical scavengers. However, individual carotenoids differ in their ability to quench singlet oxygen and scavenge for free radical species.
The carotenoids are naturally-occurring compounds that have antioxidant properties. The carotenoids are common compounds manufactured by plants or animals, and contribute greatly to the coloring of plants and animals. A number of animals, including mammals, are unable to synthesize carotenoids de novo and accordingly rely upon diet to provide carotenoid requirements. Mammals also have a limited ability to modify carotenoids. A mammal can convert .beta.-carotene to vitamin A, but most other carotenoids are deposited in mammalian tissue in unchanged form.
With respect to humans, about ten carotenoids are found in human serum. The major carotenoids in human serum are .beta.-carotene, .alpha.-carotene, cryptoxanthin, lycopene and lutein. Small amounts of zeaxanthin, phytofluene, and phytoene are found in human organs. However, of the ten carotenoids found in human serum, only two, zeaxanthin and lutein, are found in the human retina. Zeaxanthin is the predominant carotenoid in the macula and is concentrated in the cone cells in the center of the retina, i.e., the macula. Lutein is located in the peripheral retina in the rod cells. Therefore, the eye preferentially assimilates zeaxanthin over lutein in the macula and also selectively concentrates zeaxanthin, which is a more effective singlet oxygen scavenger than lutein, in the center of the eye. It has been theorized that zeaxanthin and lutein are concentrated in the retina because of their ability to quench singlet oxygen and scavenge free radicals, and thereby limit or prevent photic damage to the retina.
Therefore only two of the about ten carotenoids present in human serum are found in the retina. Beta-carotene and lycopene, the two most abundant carotenoids in human serum, either have not been detected or have been detected only in minor amounts in the retina. Beta-carotene is relatively inaccessible to the retina because .beta.-carotene is unable to cross the blood-retinal brain barrier of the retinal pigment epithelium effectively. As will be explained in detail hereinafter, small amounts of .beta.-carotene have been found to cross the blood-retinal brain barrier.
It also is known that another carotenoid, canthaxanthin, can cross the blood-retinal brain barrier and reach the retina. Canthaxanthin, like all carotenoids, is a pigment and can discolor the skin. Canthaxanthin provides a skin color that approximates a suntan, and accordingly has been used by humans to generate an artificial suntan. However, an undesirable side effect in individuals that ingested canthaxanthin at high doses for an extended time was the formation of crystalline canthaxanthin deposits in the inner layers of the retina. Therefore, the blood-retinal brain barrier of the retinal pigment epithelium permits only particular carotenoids to enter the retina. The carotenoids other than zeaxanthin and lutein that do enter the retina cause adverse effects, such as the formation of crystalline deposits by canthaxanthin, which may take several years to dissolve. Canthaxanthin in the retina also caused a decreased adaptation to the dark.
Investigators have unsuccessfully sought an antioxidant to counteract the adverse affects of singlet oxygen and free radical species on the central nervous system in general and the eye in particular. The investigators have studied the antioxidant capabilities of several compounds, including various carotenoids. Even though the carotenoids are strong antioxidants, investigators have failed to find particular carotenoids among the 600 naturally-occurring carotenoids that effectively quench singlet oxygen and scavenge for free radical species, that are capable of crossing the blood-retinal brain barrier, that do not exhibit the adverse affects of canthaxanthin after crossing the blood-retinal brain barrier, and that ameliorate central nervous system or eye disease or injury and/or retard the progression of a degenerative disease of the central nervous system or eye.
Various publications are directed to eye diseases and injuries, such as age-related macular degeneration, causes of the damage resulting from the diseases or injuries, and attempts to prevent or treat such diseases and injuries. The publications, which discuss various antioxidants, including the carotenoids and other antioxidants like .alpha.-tocopherol, include:
M. O. M. Tso, "Experiments on Visual Cells by Nature and Man: In Search of Treatment for Photoreceptor Degeneration", Investigative Ophthalmology and Visual Science, 30(12), pp. 2421-2454 (December, 1989);
W. Schalch, "Carotenoids in the Retina--A Review of Their Possible Role in Preventing or Limiting Damage Caused by Light and Oxygen", Free Radicals and Aging, I. Emerit et al. (ed.), Birkhauser Verlag, pp. 280-298 (1992);
M. O. M. Tso, "Pathogenetic Factors of Aging Macular Degeneration", Ophthalmology, 92(5), pp. 628-635 (1985);
M. Mathews-Roth, "Recent Progress in the Medical Applications of Carotenoids", Pure and Appl. Chem., 63(1), pp. 147-156 (1991);
W. Miki, "Biological Functions and Activities of Animal Carotenoids", Pure and Appl. Chem., 63(1), pp. 141-146 (1991);
M. Mathews-Roth, "Carotenoids and Cancer Prevention-Experimental and Epidemiological Studies", Pure and Appl. Chem., 57(5), pp. 717-722 (1985);
M. Mathews-Roth, "Porphyrin Photosensitization and Carotenoid Protection in Mice; In Vitro and In Vivo Studies", Photochemistry and Photobiology, 40(1), pp. 63-67 (1984);
P. DiMascio et al., "Carotenoids, Tocopherols and Thiols as Biological Singlet Molecular Oxygen Quenchers", Biochemical Society Transactions, 18, pp. 1054-1056 (1990);
T. Hiramitsu et al., "Preventative Effect of Antioxidants on Lipid Peroxidation in the Retina", Ophthalmic Res., 23, pp. 196-203 (1991);
K. Kirschfeld, "Carotenoid Pigments: Their Possible Role in Protecting Against Photooxidation in Eyes and Photoreceptor Cells", Proc. R. Soc. Lond., B216, pp. 71-85 (1982);
D. Yu et al., "Amelioration of Retinal Photic Injury by Beta-Carotene", ARVO Abstracts Invest. Ophthalmol. Vis. Sci., 28 (Suppl.), p. 7, (1987);
M. Kurashige et al., "Inhibition of Oxidative Injury of Biological Membranes by Astaxanthin", Physiol. Chem. Phys. and Med. NMR, 22, pp. 27-38 (1990); and
N. I. Krinsky et al., "Interaction of Oxygen and Oxy-radicals With Carotenoids", J. Natl. Cancer Inst., 69(1), pp. 205-210 (1982).
In general, the above-identified publications support the hypothesis that singlet oxygen and free radical species are significant contributors to central nervous system, and particularly eye, injury and disease. For example, it has reported that consumption of an antioxidant, such as ascorbic acid (Vitamin C), .alpha.-tocopherol (Vitamin E) or .beta.-carotene, can decrease the prevalence of age-related macular degeneration.
The above-identified publications also demonstrated that several carotenoids, including astaxanthin, are strong antioxidants compared to .beta.-carotene, ascorbic acid and other widely used antioxidants. The publications also relate that (1) only particular carotenoids selectively cross the blood-retinal brain barrier, and that (2) certain carotenoids other than zeaxanthin and lutein that cross the blood-retinal brain barrier cause adverse affects.
In general, the above-identified publications teach that astaxanthin is a more effective antioxidant than carotenoids such as zeaxanthin, lutein, tunaxanthin, canthaxanthin and .beta.-carotene, and than .alpha.-tocopherol. For example, the in vitro and in vivo studies disclosed in the Kurashige et al. publication with respect to astaxanthin demonstrated that the mean effective concentration of astaxanthin which inhibits lipid peroxidation was 500 times lower than that of .alpha.-tocopherol. Similarly, the Miki publication discloses that, in vitro, astaxanthin exhibits a strong quenching effect against singlet oxygen and a strong scavenging effect against free radical species.
Investigators have theorized that free radical-mediated reactions are involved in the pathogenesis of degenerative diseases, such as Alzheimer's disease, Parkinson's disease and age-related macular degeneration, ischemic/reperfusion damage and traumatic injury of the brain, spinal cord and retina, as well as in inflammatory processes of the body, and particularly the eye. This theory has been advanced by investigators examining the effectiveness of various antioxidants in ameliorating these diseases. For example, methylprednisolone, which at high doses is a strong lipid peroxidation inhibitor, has been recommended clinically for use in traumatic injury of the spinal cord.
To date, investigative efforts have been directed to preventing diseases and injury because the resulting free radical-induced damage is not effectively treatable. Therefore, a need exists for a method not only to prevent or retard, but also to ameliorate, degenerative and traumatic diseases and injuries to the central nervous system, and particularly the eye. The present invention is directed to such methods.